LILRB2/PirB mediates macrophage recruitment in fibrogenesis of nonalcoholic steatohepatitis

Inhibition of immunocyte infiltration and activation has been suggested to effectively ameliorate nonalcoholic steatohepatitis (NASH). Paired immunoglobulin-like receptor B (PirB) and its human ortholog receptor, leukocyte immunoglobulin-like receptor B (LILRB2), are immune-inhibitory receptors. However, their role in NASH pathogenesis is still unclear. Here, we demonstrate that PirB/LILRB2 regulates the migration of macrophages during NASH by binding with its ligand angiopoietin-like protein 8 (ANGPTL8). Hepatocyte-specific ANGPTL8 knockout reduces MDM infiltration and resolves lipid accumulation and fibrosis progression in the livers of NASH mice. In addition, PirB−/− bone marrow (BM) chimeras abrogate ANGPTL8-induced MDM migration to the liver. And yet, PirB ectodomain protein could ameliorate NASH by sequestering ANGPTL8. Furthermore, LILRB2-ANGPTL8 binding-promoted MDM migration and inflammatory activation are also observed in human peripheral blood monocytes. Taken together, our findings reveal the role of PirB/LILRB2 in NASH pathogenesis and identify PirB/LILRB2-ANGPTL8 signaling as a potential target for the management or treatment of NASH.

test (a and c); All the p values were two-sided and adjustments were made for multiple comparisons. kD, relative molecular weight in kilodalton; ND: normal diet, CDHFD: choline-deficient high-fat diet, and MCD, methionine-choline deficient. Source data are available as a Source Data file.

Supplementary Fig. 2 Hepatic PirB is mainly expressed in monocyte-derived macrophages (MDMs).
(a) The gating strategy for flow cytometry analyses. To discriminate KCs from MDMs, we first gated on KCs defined as CD11b lo CLEC2 hi cells. Among the remaining cells, we identified CLEC2 lo CD11b + CD64 + cells as MDMs. (b) FACS quantification of hepatic macrophages (n = 6 biologically independent samples). The data are shown as the mean ± s.e.m. and were statistically analyzed by two-tailed Student's t test. All the p values were two-sided (c) We merged PirB+ CD45+ (red) and PirB-CD45+ (blue) cells into a whole group. In the PirB+ CD45+ fraction, MDMs accounted for 43.8% (ND, left) and 77.5% (CDHFD, right). The ratio of PirB+ MDMs/PirB-MDMs increased from 1.1 to 1.5 during NASH. (d) The proportions of CD11b + , Ly6C + , and F4/80 lo cells were greater in the PirB+ CD45+ fraction than in the PirB-CD45+ fraction. ND, normal diet; CDHFD, choline-deficient high-fat diet; MDMs, monocyte-derived macrophages; KCs, Kupffer cells. Source data are available as a Source Data file.

Supplementary Fig. 3 ANGPTL8 binds to PirB on MDMs via its C-terminal domain.
(a) Coomassie blue-stained gel of the indicated protein, which was coimmunoprecipitated using anti-Flag (for ANGPTL8-flag, right) and anti-pirB (left) antibodies and identified via mass spectrometry (MS). IgG was used as a control; M, marker. (b, upper) Comparison of the amino acid sequences of ANGPTL8 in 77 mammalian species reveals high identity in 16-55 and 130-198. A, amino acid; the shades of blue indicate the degree of similarity, where dark blue indicates high similarity. (b, lower) Homology of the ANGPTL family members identifies the CC-like domain of ANGPTL8 in the A130-198 conserved region. Conservation is indicated by rectangular bars. A1-8, ANGPTL1-8; Black: signal peptide; Orange: region mediating lipoprotein lipase (LPL) binding and inhibiting its activity; Dark red: coiled-coil (CC) domain; Red: CC-like domain; Green: fibronectin-like domain (FLD). (c) Schematic showing full-length and truncated ANGPTL8. (d) Coomassie blue-stained gel of immunoprecipitation by PirB. RAW264.7 cells stably expressing truncated proteins were established by transfection with an overexpression plasmid. Red arrows show the indicated pulldown fraction; IgG was used as a control. Data shown are representative of three independent experiments with similar results (a, d). kD, relative molecular weight in kilodalton.

Supplementary Fig. 4 ANGPTL8 promotes MDM migration
(a) Representative western blot for intracellular ANGPTL8 protein expression in hepatic cells. (b) The strategy used to generate Angptl8 HepKO mice. (c) The gating strategy for flow cytometry analyses. To discriminate KCs from MDMs, we first gated on KCs defined as CD11b lo CLEC2 hi cells. Among the remaining cells, we identified CLEC2 lo CD11b+ CD64+ cells as MDMs. Finally, Ly6C+ CD11b int monocytes and Ly6C int CD11b hi neutrophils were identified in the CLEC2-CD64-fraction. (d) FACS quantification of hepatic macrophages (n = 4 biologically independent samples). (e) Experimental scheme to deplete hepatic macrophages. (f) Cell proliferation of KCs and MDMs after recombinant ANGPTL8 protein (rANGPTL8) treatment (n = 4 independent experiments). The numbers of viable cells were measured using a CCK-8 kit. (g) Blood total leukocytes in chimerism, T cells, B cells, neutrophils (Nets), and monocytes 2 weeks after bone marrow transplantation (BMT) (n = 4 biologically independent samples). (h) Quantification of MDM migration to different concentrations of rANGPTL8 over time (n = 3 or 4 independent experiments). The data are shown as the mean ± s.e.m. and were statistically analyzed by one-way ANOVA with Tukey's multiple-comparison test; All the p values were two-sided and adjustments were made for multiple comparisons. Data shown are representative of three independent experiments with similar results (a, c). kD, relative molecular weight in kilodalton. Source data are available as a Source Data file.

Supplementary Fig. 5 ANGPTL8-induced MDM activation promotes lipid accumulation in hepatocytes.
Hepatocytes were cultured in the presence or absence of MDMs and treated with or without rANGPTL8 and PA, after which cytokine levels (a, n = 4 independent experiments) from the coculture media and their corresponding mRNA expression (b, n = 3 independent experiments) of hepatocytes and MDMs were tested; TG content, TUNEL staining (c, n = 4 independent experiments), and lipogenesis genes of hepatocytes were measured (d, n = 3 independent experiments). (e) Oil Red O staining of primary hepatocytes from loxp/loxp and Angptl8 HepKO mice. Scale bar, 100 µm. The data are shown as the mean ± s.e.m. and were statistically analyzed by one-way ANOVA with Tukey's multiple-comparison test; All the p values were two-sided and adjustments were made for multiple comparisons. PA, palmitate; KCs, Kupffer cells; MDMs, monocytederived macrophages. Data shown are representative of three independent experiments with similar results (e). Source data are available as a Source Data file. Supplementary Fig. 6 PirB and its downstream signalling molecules mediate ANGPTL8-induced cytokine production and the migration of MDMs.
(a) Quantification of flag-tagged (red) rA8 on MDMs pretreated with 1 µg/ml indicated antibodies at different rA8 concentrations (n = 3 independent experiments). IgG was used as a control. (b-e) mRNA expression of PirA/PirB (b) and cytokines (c-e) in RAW264.7 cells transfected with lentiviruses containing shRNA (n = 9 cells examined over 3 independent experiments) of scramble, PirA (n = 3 or 5 cells examined over 3 independent experiments) and PirB (n = 9 cells examined over 3 independent experiments). (f-h) Cytokine expression in MDMs pretreated with phosphorylation inhibitors before ANGPTL8 treatment (n = 6 or 9 cells examined over 3 independent experiments). The data are shown as the mean ± s.e.m. and were statistically analyzed by one-way ANOVA with Tukey's multiple-comparison test; All the p values were two-sided and adjustments were made for multiple comparisons. MDMs, monocyte-derived macrophages; rANGPTL8 (rA8), recombinant ANGPTL8 protein. Source data are available as a Source Data file. as the mean ± s.e.m. and were statistically analyzed by two-tailed Student's t-test (a and b) or one-way ANOVA with Tukey's multiple-comparison test (d-i); All the p values were two-sided and adjustments were made for multiple comparisons. Data shown are representative of three independent experiments with similar results (c, d, l). kD, relative molecular weight in kilodalton; sPirB, recombinant soluble PirB ectodomain protein; ND: normal diet; CDHFD: choline-deficient high-fat diet. Source data are available as a Source Data file. . The data are shown as the mean ± s.e.m. and were statistically analyzed by two-tailed Student's t test (b, d) or one-way ANOVA with Tukey's multiple-comparison test (e); All the p values were two-sided and adjustments were made for multiple comparisons. Data shown are representative of three independent experiments with similar results (a). hMDMs, human monocyte-derived macrophages. Source data are available as a Source Data file.

Recombinant proteins
All the recombinant proteins were tested by SDS-PAGE quantitative densitometry by Coomassie blue staining, and the purities were 95%-98%. No aggregated or sticky protein was found. Proteins were reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, and were added 5% glycerol (final concentration) and aliquoting for long-term storage at -20℃/-80℃.